Cytochrome P450 (P450) monooxygenases are ubiquitous hemoproteins present in microorganisms, plants and animals. Comprised of a large and diverse group of isozymes, P450s mediate a great array of oxidative reactions using a wide range of compounds as substrates, and including reactions involved in biosynthetic processes such as phenylpropanoid, fatty acid, and terpenoid biosynthesis; metabolism of natural products; and detoxification of foreign substances (xenobiotics). See e.g., Schuler (1996) Crit. Rev. Plant Sci. 15:235-284 (1996). In a typical P450 catalyzed reaction, one atom of molecular oxygen (O2) is incorporated into the substrate, and the other atom is reduced to water. Electrons are supplied through oxidation of NADPH. For most eukaryotic P450s, NADPH:cytochrome P450 reductase, a membrane-bound flavoprotein, transfers the necessary two electrons from NADPH to the cytochrome P450 (Bolwell et al (1994) Phytochemistry 37: 1491-1506; Mizutani and Ohta (1998) Plant Physiol., 16, 357-367).
Plant cytochrome P450s are known to be involved in the metabolism and detoxification of numerous pesticides as well as in the biosynthesis of primary and secondary metabolites. Much of the evidence has been gathered via traditional chemistry techniques (Shuler (1996) Crit. Rev. Plant. Sci. 15:235-284; Bolwell et al. (1994) Phytochemistry 37:1491-1506; Frear et al. (1969) Phytochemistry 8:2157-2169) and through studies of mammalian or bacterial genes in plants (Shiota et al. (1994) Plant Physiol. 106:17-23; O'Keefe et al. (1994) Plant Physiol. 105:473-482).
Recently, endogenous plant P450s have been successfully cloned, expressed, characterised and assayed in heterologous systems (Siminszky et al. (1999) PNAS (USA) 96:1750-1755). For example, CYP76B1 was cloned (Robineau et al. (1998) Plant Physiol., 118:1049-1056) from the Jerusalem artichoke (Helianthus tuberosus). This xenobiotic inducible cytochrome P450 was found to be strongly inducible and to catalyze the rapid oxidative N-dealkylation of various phenylurea herbicides to yield non-phytotoxic metabolites. Heterologous expression of the CYP76B1 gene in tobacco (Nicotiana tabacum) and Arabidopsis resulted in increased rates of herbicide oxidation and was, by itself, sufficient to yield a 20 fold level of tolerance to linuron, a compound detoxified by a single dealkylation, and also a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification (Didierjean et al. (2002) Plant Physiol. 130:179-189).
Frear et al. (1969) Phytochemistry 8:2157-2169 demonstrated the metabolism of monuron by a mixed-function oxidase located in a microsomal fraction of cotton seedlings. Further evidence has accumulated supporting the involvement of P450s in the metabolism and detoxification of numerous herbicides representing several distinct classes of compounds (reviewed in Bolwell et al. (1994) Phytochemistry 37:1491-1506; Shuler (1996) Crit. Rev. Plant. Sci. 15:235-284). Likewise, the crop/weed selectivity of certain HPPD-inhibiting herbicides such as mesotrione (Hawkes et al. (2001) Proc. Brighton Pest Cont Conf-Weeds, BCPC, 563-568) and topramezone (Grossmann and Ehrhardt (2007) Pest Mgmt. Sci. 63:429-439) for example is largely dependent on the activity of cytochrome P450 type enzymes.
Differential herbicide metabolizing P450 activities are believed to represent one of the mechanisms that enable certain crop species to be more tolerant of a particular herbicide than other crop or weedy species. The following patents and applications collect information useful for the background understanding of cytochrome P450 use for herbicide tolerance (U.S. Pat. Nos. 6,380,465; 6,121,512; 5,349,127; and PCT Patent App. Pub. No. WO2007000077). See also U.S. Pat. Nos. 6,649,814 and 6,300,544 for disclosure relating to cytochrome P450 monooxygenases for obtaining transgenic plants resistant to insects, acarids, or nematodes, or with improved nutritive value.
Methods for providing plants which are tolerant to HPPD herbicides which comprise transformation of plant material with polynucleotides comprising regions which encode HPPD enzymes are known (See, e.g., U.S. Application Publication Number 2004/0058427; PCT application WO98/20144; PCT application WO 02/46387; see also U.S. Application Publication Number 2005/0246800 relating to identification and labelling of soybean varieties as being relatively HPPD tolerant). However what has not hitherto been generally recognised is that cytochrome P450 enzymes can provide additional or commercial levels of tolerance to HPPD-inhibitor and other herbicides, when expressed either in combination with HPPD enzymes, or in tolerant backgrounds, or alone in a transformed plant. While a given HPPD enzyme may provide a useful level of tolerance to some HPPD-inhibitor herbicides it may be quite inadequate to provide commercial levels of tolerance to a different, more desirable HPPD-inhibitor herbicide which, for example, may control a different spectrum of weeds, be cheaper to make or offer environmental benefits. As well as particular HPPD enzymes and the polynucleotides which encode them the current invention provides a means of enhancing the effect of HPPD enzymes suitable for providing commercially useful levels of resistance to particular HPPD-inhibitor herbicide chemistries through the use of cytochrome P450 enzymes that degrade certain, below-specified, HPPD-inhibitor herbicide chemistries. In addition, expression of these cytochrome P450 enzymes also provides a means of reducing the amount of parent active herbicide that persists in plant tissues and of therefore decreasing the overall amount of parent herbicide residue entering the food and feed chain as a result of application to food or feed crops.
In addition, the genes disclosed herein also provide an alternative process for providing resistance to various xenobiotics including herbicides and including some types, such as sulfonylureas having a non-HPPD mode of action.